Method of making a microbead array with attached biomolecules

ABSTRACT

A method and apparatus for the manipulation of colloidal particulates and biomolecules at the interface between an insulating electrode such as silicon oxide and an electrolyte solution. Light-controlled electrokinetic assembly of particles near surfaces relics on the combination of three functional elements: the AC electric field-induced assembly of planar aggregates; the patterning of the electrolyte/silicon oxide/silicon interface to exert spatial control over the assembly process; and the real-time control of the assembly process via external illumination. The present invention provides a set of fundamental operations enabling interactive control over the creation and placement of planar arrays of several types of particles and biomolecules and the manipulation of array shape and size. The present invention enables sample preparation and handling for diagnostic assays and biochemical analysis in an array format, and the functional integration of these operations. In addition, the present invention provides a procedure for the creation of material surfaces with desired properties and for the fabrication of surface-mounted optical components.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/664,953 filed Oct. 31, 2012, (now U.S. Pat. No. 8,691,594) which is acontinuation of U.S. application Ser. No. 11/760,814, filed Jun. 11,2007 (now U.S. Pat. No. 8,309,368), which is a continuation of U.S.application Ser. No. 10/910,466, filed Aug. 3, 2004 (now U.S. Pat. No.7,427,512), which is a continuation of U.S. application Ser. No.09/690,040, filed Oct. 17, 2000 (now U.S. Pat. No. 6,797,524), which isa continuation of U.S. application Ser. No. 09/171,550, filed Oct. 26,1998 (now U.S. Pat. No. 6,251,691), which is a 35 U.S.C. Section 371national stage entry of PCT/US97/08159, filed Apr. 24, 1997, whichclaims priority to U.S. Provisional Application No. 60/016,642, filedApr. 25, 1996. All of the applications and patents cited above arehereby incorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention generally relates to the field of materialsscience and analytical chemistry.

The present invention specifically relates to the realization of acomplete, functionally integrated system for the implementation ofbiochemical analysis in a planar, miniaturized format on the surface ofa conductive and/or photoconductive substrate, with applications inpharmaceutical and agricultural drug discovery and in in-vitro orgenomic diagnostics. In addition, the method and apparatus of thepresent invention may be used to create material surfaces exhibitingdesirable topographical relief and chemical functionality, and tofabricate surface-mounted optical elements such as lens arrays.

BACKGROUND OF THE INVENTION

I—Ions, Electric Fields, and Fluid Flow: Field-Induced Formation ofPlanar Bead Arrays

Electrokinesis refers to a class of phenomena elicited by the action ofan electric field on the mobile ions surrounding charged objects in anelectrolyte solution. When an object of given surface charge is immersedin a solution containing ions, a diffuse ion cloud forms to screen theobject's surface charge. This arrangement of a layer of (immobile)charges associated with an immersed object and the screening cloud of(mobile) counterions in solution is referred to as a “double layer”. Inthis region of small but finite thickness, the fluid is notelectroneutral. Consequently, electric fields acting on this region willset in motion ions in the diffuse layer, and these will in turn entrainthe surrounding fluid. The resulting flow fields reflect the spatialdistribution of ionic current in the fluid. Electroosmosis representsthe simplest example of an electrokinetic phenomenon. It arises when anelectric field is applied parallel to the surface of a sample containeror electrode exhibiting fixed surface charges, as in the case of asilicon oxide electrode (in the range of neutral pH). As counterions inthe electrode double layer are accelerated by the electric field, theydrag along solvent molecules and set up bulk fluid flow. This effect canbe very substantial in narrow capillaries and may be used to advantageto devise fluid pumping systems.

Electrophoresis is a related phenomenon which refers to thefield-induced transport of charged particles immersed in an electrolyte.As with electroosmosis, an electric field accelerates mobile ions in thedouble layer of the particle. If, in contrast to the earlier case, theparticle itself is mobile, it will compensate for this field-inducedmotion of ions (and the resulting ionic current) by moving in theopposite direction. Electrophoresis plays an important role inindustrial coating processes and, along with electroosmosis, it is ofparticular interest in connection with the development of capillaryelectrophoresis into a mainstay of modern bioanalytical separationtechnology.

In confined geometries, such as that of a shallow experimental chamberin the form of a “sandwich” of two planar electrodes, the surface chargedistribution and topography of the bounding electrode surfaces play aparticularly important role in determining the nature and spatialstructure of electroosmotic flow. Such a “sandwich” electrochemical cellmay be formed by a pair of electrodes separated by a shallow gap.Typically, the bottom electrode will be formed by an oxide-cappedsilicon wafer, while the other electrode is formed by opticallytransparent, conducting indium tin oxide (ITO). The silicon (Si) waferrepresents a thin slice of a single crystal of silicon which is doped toattain suitable levels of electrical conductivity and insulated from theelectrolyte solution by a thin layer of silicon oxide (SiOx).

The reversible aggregation of beads into planar aggregates adjacent toan electrode surface may be induced by a (DC or AC) electric field thatis applied normal to the electrode surface. While the phenomenon hasbeen previously observed in a cell formed by a pair of conductive ITOelectrodes (Richetti, Prost and Barois, J. Physique Lettr. 45, L-1137through L-1143 (1984)), the contents of which are incorporated herein byreference, it has been only recently demonstrated that the underlyingattractive interaction between beads is mediated by electrokinetic flow(Yeh, Seul and Shraiman, “Assembly of Ordered Colloidal Aggregates byElectric Field Induced Fluid Flow”, Nature 386, 57-59 (1997), thecontents of which are incorporated herein by reference. This flowreflects the action of lateral non-uniformities in the spatialdistribution of the current in the vicinity of the electrode. In thesimplest case, such non-uniformities are introduced by the very presenceof a colloidal bead near the electrode as a result of the fact that eachbead interferes with the motion of ions in the electrolyte. Thus, it hasbeen observed that an individual bead, when placed near the electrodesurface, generates a toroidal flow of fluid centered on the bead.Spatial non-uniformities in the properties of the electrode can also beintroduced deliberately by several methods to produce lateral fluid flowtoward regions of low impedance. These methods are described insubsequent sections below.

Particles embedded in the electrokinetic flow are adverted regardless oftheir specific chemical or biological nature, while simultaneouslyaltering the flow field. As a result, the electric field-inducedassembly of planar aggregates and arrays applies to such diverseparticles as: colloidal polymer lattices (“latex beads”), lipidvesicles, whole chromosomes, cells and biomolecules including proteinsand DNA, as well as metal or semiconductor colloids and clusters.

Important for the applications to be described is the fact that theflow-mediated attractive interaction between beads extends to distancesfar exceeding the characteristic bead dimension. Planar aggregates areformed in response to an externally applied electric field anddisassemble when the field is removed. The strength of the applied fielddetermines the strength of the attractive interaction that underlies thearray assembly process and thereby selects the specific arrangementadopted by the beads within the array. That is, as a function ofincreasing applied voltage, beads first form planar aggregates in whichparticles are mobile and loosely packed, then assume a tighter packing,and finally exhibit a spatial arrangement in the form of a crystalline,or ordered, array resembling a raft of bubbles. The sequence oftransitions between states of increasing internal order is reversible,including complete disassembly of planar aggregates when the appliedvoltage is removed. In another arrangement, at low initialconcentration, beads form small clusters which in turn assume positionswithin an ordered “superstructure”.

II—Patterning of Silicon Oxide Electrode Surfaces

Electrode patterning in accordance with a predetermined designfacilitates the quasi-permanent modification of the electrical impedanceof the EIS (Electrolyte-Insulator-Semiconductor) structure of interesthere. By spatially modulating the EIS impedance, electrode-patterningdetermines the ionic current in the vicinity of the electrode. Dependingon the frequency of the applied electric field, beads either seek out,or avoid, regions of high ionic current. Spatial patterning thereforeconveys explicit external control over the placement and shape of beadarrays.

While patterning may be achieved in many ways, two procedures offerparticular advantages. First, UV-mediated re-growth of a thin oxidelayer on a properly prepared silicon surface is a convenient methodologythat avoids photolithographic resist patterning and etching. In thepresence of oxygen, UV illumination mediates the conversion of exposedsilicon into oxide. Specifically, the thickness of the oxide layerdepends on the exposure time and may thus be spatially modulated byplacing patterned masks into the UV illumination path. This modulationin thickness, with typical variations of approximately 10 Angstroms,translates into spatial modulations in the impedance of the Si/SiOxinterface while leaving a flat and chemically homogeneous top surfaceexposed to the electrolyte solution. Second, spatial modulations in thedistribution of the electrode surface charge may be produced byUV-mediated photochemical oxidation of a suitable chemical species thatis first deposited as a monolayer film on the SiOx surface. This methodpermits fine control over local features of the electrode double layerand thus over the electrokinetic flow.

A variation of this photochemical modulation is the creation of lateralgradients in the EIS impedance and hence in the current generated inresponse to the applied electric field. For example, this is readilyaccomplished by controlling the UV exposure so as to introduce a slowlateral variation in the oxide thickness or in the surface chargedensity. As discussed below, control over lateral gradients serves toinduce lateral bead transport and facilitates the implementation of suchfundamental operations as capturing and channeling of beads to apredetermined destination along conduits in the form of impedancefeatures embedded in the Si/SiOx interface. Photochemical patterning offunctionalized chemical overlayers also applies to other types ofelectrode surfaces including ITO.

III—Light-Controlled Modulation of the Interfacial Impedance

The spatial and temporal modulation of the EIS-impedance in accordancewith a pattern of external illumination provides the basis to controlthe electrokinetic forces that mediate bead aggregation. Thelight-modulated electrokinetic assembly of planar colloidal arraysfacilitates remote interactive control over the formation, placement andrearrangement of bead arrays in response to corresponding illuminationpatterns and thereby offers a wide range of interactive manipulations ofcolloidal beads and biomolecules.

To understand the principle of this methodology, it will be helpful tobriefly review pertinent photoelectric properties of semiconductors, ormore specifically, those of the EIS structure formed by the Electrolytesolution (E), the Insulating SiOx layer (I) and the Semiconductor (S).The photoelectric characteristics of this structure are closely relatedto those of a standard Metal-Insulator-Semiconductor (MIS) orMetal-Oxide-Semiconductor (MOS) devices which are described in S. M.Sze, “The Physics of Semiconductors”, 2nd Edition, Chapt. 7 (WileyInterscience 1981), the contents of which art incorporated herein byreference.

The interface between the semiconductor and the insulating oxide layerdeserves special attention. Crucial to the understanding of theelectrical response of the MOS structure to light is the concept of aspace charge region of small but finite thickness that forms at theSi/SiOx interface in the presence of a bias potential. In the case ofthe EIS structure, an effective bias, in the form of a junctionpotential, is present under all but very special conditions. The spacecharge region forms in response to the distortion of the semiconductor'svalence and conduction bands (“band bending”) in the vicinity of theinterface. This condition in turn reflects the fact that, while there isa bias potential across the interface, there is ideally no chargetransfer in the presence of the insulating oxide. That is, inelectrochemical language, the EIS structure eliminates Faradaic effects.Instead, charges of opposite sign accumulate on either side of theinsulating oxide layer and generate a finite polarization.

In the presence of a reverse bias, the valence and conduction band edgesof an n-doped semiconductor bend upward near the Si/SiOx interface andelections flow out of the interfacial region in response to thecorresponding potential gradient. As a result, a majority carrierdepletion layer is formed in the vicinity of the Si/SiOx interface.Light absorption in the semiconductor provides a mechanism to createelectron-hole pairs within this region. Provided that they do notinstantaneously recombine, electron-hole pairs are split by the locallyacting electric field, and a corresponding photocurrent flows. It isthis latter effect that affords control over the electrokinetic assemblyof beads in the electrolyte solution.

To understand in more detail the pertinent frequency dependence of thelight-induced modulation of the EIS impedance, two aspects of theequivalent circuit representing the EIS structure are noteworthy. First,there are close analogies between the detailed electricalcharacteristics of the electric double layer at the electrolyte-oxideinterface, and the depletion layer at the interface between thesemiconductor and the insulator. As with the double layer, the depletionlayer exhibits electrical characteristics similar to those of acapacitor with a voltage-dependent capacitance. As discussed,illumination serves to lower the impedance of the depletion layer.Second, given its capacitive electrical response, the oxide layer willpass current only above a characteristic (“threshold”) frequency.Consequently, provided that the frequency of the applied voltage exceedsthe threshold, illumination can lower the effective impedance of theentire EIS structure.

This effective reduction of the EIS impedance also depends on the lightintensity which determines the rate of generation of electron-holepairs. In the absence of significant recombination, the majority ofphotogenerated electrons flow out of the depletion region and contributeto the photocurrent. The remaining hole charge accumulates near theSi/SiOx interface and screens the electric field acting in the depletionregion. As a result, the rate of recombination increases, and theefficiency of electron-hole separation, and hence the photocurrent,decreases. For given values of frequency and amplitude of the appliedvoltage, one therefore expects that as the illumination intensityincreases, the current initially increases to a maximum level and thendecreases. Similarly, the impedance initially decreases to a minimumvalue (at maximum current) and then decreases.

This intensity may be used to advantage to induce the lateraldisplacement of beads between fully exposed and partially masked regionsof the interface. As the illumination intensity is increased, the fullyexposed regions will correspond to the regions of interface of lowestimpedance, and hence of highest current, and beads will be drawn intothese regions. As the fully exposed regions reach the state ofdecreasing photocurrent, the effective EIS impedance in those regionsmay exceed that of partially masked regions, with a resulting inversionof the lateral gradient in current. Beads will then be drawn out of thefully exposed regions. Additionally, time-varying changes in theillumination pattern may be used to effect bead motion.

IV—Integration of Biochemical Analysis in a Miniaturized, Planar Format

The implementation of assays in a planar array format, particularly inthe context of biomolecular screening and medical diagnostics, has theadvantage of a high degree of parallelity and automation so as torealize high throughput in complex, multi-step analytical protocols.Miniaturization will result in a decrease in pertinent mixing timesreflecting the small spatial scale, as well as in a reduction ofrequisite sample and reagent volumes as well as power requirements. Theintegration of biochemical analytical techniques into a miniaturizedsystem on the surface of a planar substrate (“chip”) would yieldsubstantial improvements in the performance, and reduction in cost, ofanalytical and diagnostic procedures.

Within the context of DNA manipulation and analysis, initial steps havebeen taken in this direction (i.e., miniaturization) by combining on aglass substrate, the restriction enzyme treatment of DNA and thesubsequent separation of enzyme digests by capillary electrophoresis,see, for example, Ramsey, PCT Publication No. WO 96/04547, the contentsof which are incorporated herein by reference, or the amplification ofDNA sequences by application of the polymerase chain reaction (PCR) withsubsequent electrophoretic separation, see, for example, U.S. Pat. Nos.5,498,392 and 5,587,128 to Wilding et al., the contents of which areincorporated herein by reference.

While these standard laboratory processes have been demonstrated in aminiaturized format, they have not been used to form a complete system.A complete system will require additional manipulation such as front-endsample processing, binding and functional assays and the detection ofsmall signals followed by information processing. The true challenge isthat of complete functional integration because it is here that systemarchitecture and design constraints on individual components willmanifest themselves. For example, a fluidic process is required toconcatenate analytical steps that require the spatial separation, andsubsequent transport to new locations, of sets of analyte. Severalpossibilities have been considered including electroosmotic pumping andtransport of droplets by temperature-induced gradients in local surfacetension. While feasible in demonstration experiments, these techniquesplace rather severe requirements on the overall systems lay-out tohandle the very considerable DC voltages required for efficientelectroosmotic mixing or to restrict substrate heating when generatingthermally generated surface tension gradients so as to avoid adverseeffects on protein and other samples.

SUMMARY OF THE INVENTION

The present invention combines three separate functional elements toprovide a method and apparatus facilitating the real-time, interactivespatial manipulation of colloidal particles (“beads”) and molecules atan interface between a light sensitive electrode and an electrolytesolution. The three functional elements are: the electric field-inducedassembly of planar particle arrays at an interface between an insulatingor a conductive electrode and an electrolyte solution; the spatialmodulation of the interfacial impedance by means of UV-mediated oxideregrowth or surface-chemical patterning; and, finally, the real-time,interactive control over the state of the interfacial impedance bylight. The capabilities of the present invention originate in the factthat the spatial distribution of ionic currents, and thus the fluid flowmediating the array assembly, may be adjusted by external intervention.Of particular interest is the introduction of spatial non-uniformitiesin the properties of the pertinent EIS stricture. As described herein,such inhomogeneities, either permanent or temporary in nature, may beproduced by taking advantage of the physical and chemical properties ofthe EIS structure.

The invention relates to the realization of a complete, functionallyintegrated system for the implementation of biochemical analysis in aplanar, miniaturized format on the surface of a silicon wafer or similarsubstrate. In addition, the method and apparatus of the presentinvention may be used to create material surfaces exhibiting desirabletopographical relief and chemical functionality, and to fabricatesurface-mounted optical elements such as lens arrays.

The combination of three functional elements endows the presentinvention with a set of operational capabilities to manipulate beads andbead arrays in a planar geometry to allow the implementation ofbiochemical analytical techniques. These fundamental operations apply toaggregates and arrays of particles such as: colloidal polymer lattices,vesicles, whole chromosomes, cells and biomolecules including proteinsand DNA, as well as metal or semiconductor colloids and clusters.

Sets of colloidal particles may be captured, and arrays may be formed indesignated areas on the electrode surface (FIGS. 1a, 1b and FIGS. 2a-d). Particles, and the arrays they form in response to the applied field,may be channeled along conduits of any configuration that are eitherembedded in the Si/SiOx interface by UV-oxide patterning or delineatedby an external pattern of illumination. This channeling (FIGS. 1c, 1d,1e , FIGS. 3c, 3d ), in a direction normal to that of the appliedelectric field, relies on lateral gradients in the impedance of the EISstructure and hence in the field-induced current. As discussed herein,such gradients may be introduced by appropriate patterns ofillumination, and this provides the means to implement a gated versionof translocation (FIG. 1e ). The electroltinetic flow mediating thearray assembly process may also be exploited for the alignment ofelongated particles, such as DNA, near the surface of the electrode. Inaddition, the present invention permits the realization of methods tosort and separate particles.

Arrays of colloidal particles may be placed in designated areas andconfined there until released or disassembled. The overall shape of thearray may be delineated by UV-oxide patterning or, in real time, byshaping the pattern of illumination. This capability enables thedefinition of functionally distinct compartments, permanent ortemporary, on the electrode surface. Arrays may be subjected to changesof shape imposed in real time, and they may be merged with other ways(FIG. 1f ) or split into two or more subarrays or clusters (FIG. 1g ,FIGS. 4a, 4b ). In addition, the local state of order of the array aswell as the lateral particle density may be reversibly adjusted by wayof the external electric field or modified by addition of a second,chemically inert bead component.

The present invention also allows for the combination of fundamentaloperations to develop increasingly complex products and processes.Examples given herein describe the implementation of analyticalprocedures essential to a wide range of problems in materials science,pharmaceutical drug discovery, genomic mapping and sequencingtechnology. Important to the integration of these and otherfunctionalities in a planar geometry is the capability, provided by thepresent invention, to impose temporary or permanent compartmentalizationin order to spatially isolate concurrent processes or sequential stepsin a protocol and the ability to manipulate sets of particles in amanner permitting the concatenation of analytical procedures that areperformed in different designated areas on the substrate surfaces.

BRIEF DESCRIPTION OF DRAWINGS

Other objects, features and advantages of the invention discussed in theabove brief explanation will be more clearly understood when takentogether with the following detailed description of an embodiment whichwill be understood as being illustrative only, and the accompanyingdrawings reflecting aspects of that embodiment, in which:

FIGS. 1A-1H are illustrations of the fundamental operations for beadmanipulation;

FIGS. 2A and 2B are photographs illustrating the process of capturingparticles in designated areas on the substrate surface;

FIGS. 2C and 2D are photographs illustrating the process of excludingparticles from designated areas on the substrate surface;

FIGS. 3A and 3B are illustrations of the oxide profile of an Si/SiOxelectrode;

FIGS. 3C and 3D are photographs of the channeling of particles alongconduits;

FIGS. 4A and 4B are photographs of the splitting of an existingaggregate into small clusters;

FIG. 5 is a photograph of the lensing action of individual colloidalbeads;

FIGS. 6A-6C are side view illustrations of a layout-preserving transferprocess from a microtiter plate to a planar cell;

FIG. 7 is a photograph of the inclusion of spacer particles within beadclusters;

FIG. 8 is an illustration of binding assay variations;

FIGS. 9A and 9B are illustrations of two mechanisms of particle sorting;

FIG. 10 is an illustration of a planar array of bead-anchoredprobe-target complexes; and

FIGS. 11A-11E are illustrations of DNA stretching in accordance with thepresent invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The three functional elements of the present invention may be combinedso as to provide a set of fundamental operations for the interactivespatial manipulation of colloidal particles and molecules, assembledinto planar aggregates adjacent to an electrode surface. In thefollowing description, fundamental operations in this “toolset” aredescribed in order of increasing complexity. Specifically, it is usefulto adopt a classification scheme based on the total number of inputs andoutputs, or “terminals”, involved in a given operation. For example, themerging of two separate arrays, or sets of particles, into one would bea “three-terminal” operation, involving two inputs and one output. Theconverse three-terminal operation, involving one input and two outputs,is the splitting of a given array into two subarrays.

Experimental conditions yielding the phenomena depicted in the variousphotographs included herein are as follows. An electrochemical cell isformed by a pair of planar ITO electrodes, composed of an ITO layerdeposited on a glass substrate, or by a Si/SiOx electrode on the bottomand an ITO electrode on the top, separated by a typical gap of 50microns or less. Given its dependence on the photoelectric properties ofthe Si/SiOx interface, light control is predicated on the use of aSi/SiOx electrode. Leads, in the form of platinum wires, are attached tothe ITO and to the silicon electrode, which is first etched to removethe insulating oxide in the contact region, by means of silver epoxy.The cell is first assembled and then filled, relying on capillaryaction, with a suspension of colloidal beads, 1 or 2 microns indiameter, at a typical concentration of 0.1% solids in 0.1 mM azidesolution, corresponding to approximately 2×10^8 particles permilliliter. The number is chosen so as to yield between ½ and 1 fullmonolayer of particles on the electrode surface. Anionic (e.g.,carboxylated polystyrene, cationic (e.g., aminated polystyrene) ornominally neutral (e.g., polystyrene) have all been used to demonstratethe basic phenomena underlying the three functional elements of thepresent invention. The silicon electrode was fabricated from a 1inch-square portion of a Si (100) wafer, typically 200-250 micronsthick, n-doped to typically 0.01 Ohm cm resistivity, and capped with athin oxide of typically 30-40 Angstroms thickness. A thick oxide layerof typically 6000-8000 Angstrom thickness, grown under standardconditions in a furnace at 950 degrees C., may be etched by standardphotolithography to define the structures of interest. Alternatively, athin oxide layer may be regrown on a previously stripped surface of(100)-orientation under UV illumination. Given its ease ofimplementation and execution, UV-mediated oxide regrowth is thepreferable technique: it provides the means to pattern the surface byplacing a quartz mask representing the desired pattern in the path of UVillumination and it leaves a chemically homogeneous, topographicallyflat top surface. To avoid non-specific particle adsorption to theelectrode surface, stringent conditions of cleanliness should befollowed, such as those set forth in the General Experimental Conditionsbelow.

The fundamental one-terminal operation is a “capture-and-hold” operation(FIG. 1a ) which forms an array of particles in a designated area ofarbitrary outline on the surface that is delineated by UV-mediated oxidepatterning or by a corresponding pattern of illumination projected on anotherwise uniform Si/SiOx substrate surface. FIGS. 2a and 2b illustratebead capture on a surface characterized by a very thin oxide region 22(approximately 20-30 Angstroms in thickness) and correspondingly lowimpedance, while the remaining surface is covered with the original,thick oxide with correspondingly high impedance. In FIG. 2a , there isno applied field, and hence, no bead capture. In contrast, in FIG. 2b ,an electric field is applied (10 Vp-p source, 1 kHz) and bead captureoccurs within the thin oxide region 22. Under these conditions, an arraystarts to grow within less than a second and continues to grow over thenext approximately 10 seconds as beads arrive from increasingly largerdistances to add to the outward growing perimeter of region 22. Growthstops when the array approaches the outer limit of the delineated targetarea, i.e., the area defined by the thin oxide having a low impedance.The internal state of order of the captured aggregate of beads isdetermined by the strength of the applied voltage, higher valuesfavoring increasingly denser packing of beads and the eventual formationof ordered arrays displaying a hexagonally crystalline configuration inthe form of a bubble raft. The array remains in place as long as theapplied voltage is present. Removal of the applied voltage results inthe disassembly of the array.

The “capture-and-hold” operation may also be implemented underillumination with visible or infrared light, for example by simplyprojecting a mask patterned with the desired layout onto the Si/SiOxelectrode. A regular 100 W quartz microscope illuminator has been usedfor this purpose on a Zeiss UEM microscope, with apertures or masksinserted in the intermediate image plane to provide the required shapein the plane of the electrode (when focused properly under conditions ofKoehler illumination). Alternatively, an IR laser diode with output of 3mW at 650-680 nm also has been used. The use of external illuminationrather than oxide patterning for the spatial confinement of particlesallows the confinement pattern to be easily modified.

Related to “capture-and-hold” is the one-terminal operation of“exclude-and-hold” (FIG. 1b ) which clears particles from a designatedarea on the surface. Increasing the frequency of the applied voltage toapproximately 100 kHz leads to an inversion in the preference ofparticles which assemble is the thin-oxide portion of the surface (e.g.,region 22, FIG. 2b ) and instead form structures decorating the outsideof the target area perimeter. Rather than relying on this effect, theexclusion of particles from the desired areas is also accomplished, inanalogy to the original “capture-and-hold” operations, by simplyembedding the corresponding structure in the Si/SiOx interface byUV-mediated oxide regrowth. In the example of FIGS. 2e and 2d , this isachieved, under conditions otherwise identical to those described above,with respect to FIGS. 2a and 2b , by applying 20V (pp) at 10 kHz. Whilethe oxide thickness in the non designated areas 24 is approximately 30Angstroms, the value in the designated square areas 26 is approximately40 Angstroms, implying a correspondingly higher impedance at the appliedfrequency.

The “capture-and-hold” operation enables the spatialcompartmentalization of the substrate surface into functionally distinctregions. For example, particles of distinct chemical type, introducedinto the electrochemical cell at different times or injected indifferent locations, can be kept in spatially isolated locations byutilizing this operation.

The fundamental two-terminal operation is translocation (FIG. 1c ), orthe controlled transport of a set of particles from location O tolocation F on the surface; here, O and F are target areas to which theabove-described one-terminal operations may be applied. Theone-dimensional, lateral bead transport used in translocation isachieved by imposing a lateral current along a conduit connecting areasO and F, as shown in FIGS. 3a and 3b or by projecting a correspondinglinear pattern of illumination. In this channeling operation, beads movein the direction of lower impedance in the direction of the arrow shownin FIGS. 3a and 3b , in accordance with the underlying electrokineticflow.

Oxide patterning may be utilized in two ways to create a lateral currentalong the Si/SiOx interface. The simplest method is depicted in FIG. 3cand shows a large open holding area 32 fed by three narrow conduits 34defined by etching a thermal oxide. Beads move to the holding area 32along the narrow conduits 34 to form a bead array. FIG. 3d is a largescale view of the array of FIG. 3c . The principle invoked in creatingtransport is that of changing the aspect ratio (narrow conduit connectedto wide holding area) of the embedded pattern with constant values ofthin oxide thickness inside and thick oxide outside, as illustrated inFIG. 3a . In FIGS. 3c and 3d , the applied voltage was 10V (pp) at 10kHz. An alternative approach for creating bead transport, enabled byUV-mediated oxide regrowth, is to vary the oxide thickness along theconduit in a controlled fashion. This is readily accomplished by UVexposure through a graduated filter. Differences in the oxide thicknessbetween O and F of as little as 5-10 Angstroms suffice to effect lateraltransport. In this situation, the aspect ratio of the conduit andholding areas need not be altered. This is illustrated in FIG. 3 b.

The use of external illumination to define conduits, by varying theillumination intensity along the conduit to create the requisiteimpedance gradient, has the advantage that the conduit is only atemporary structure, and that the direction of motion may be modified orreversed if so desired. The present invention provides for mechanisms oflight-mediated active linear transport of planar aggregates of beadsunder interactive control. This is achieved by adjusting an externalpattern of illumination in real time, either by moving the patternacross the substrate surface in such a way as to entrain the illuminatedbead array or by electronically modulating the shape of the pattern toinduce motion of particles.

Two modes of light-mediated, active transport are:

i) Direct Translocation (“tractor beam”) which is a method oftranslocating arrays and of delineating their overall shape by adjustingparameters so as to favor particle assembly within illuminated areas ofthe surface, as described herein. Arrays simply follow the imposedpattern. The rate of motion is limited by the mobility of particles inthe fluid and thus depends on particle diameter and fluid viscosity.

ii) Transverse Array Constriction is a bead transport mechanism relatedto peristaltic pumping of fluids through flexible tubing. Thelight-control component of the present invention may be used for asimple implementation of this very general concept. A multi-componentplanar aggregate of beads is confined to a rectangular channel, byUV-patterning if so desired, or simply by light. Beads are free to movealong the channel by diffusion (in either direction). An illuminationpattern matching the transverse channel dimension is set up and is thenvaried in time so as to produce a transverse constriction wave thattravels in one direction along the channel. Such a constriction wave maybe set up in several ways. A conceptually simple method is to project aconstricting mask onto the sample and move the projected mask pattern inthe desired fashion. This method also may be implemented electronicallyby controlling the illumination pattern of a suitable array of lightsources, thus obviating the need for moving parts in the optical train.

The control of lateral bead transport by changing or moving patterns ofillumination has the advantage that it may be applied whenever andwherever (on a given substrate surface) required, without the need toimpose gradients in impedance by predefined UV patterning. On the otherhand, a predefined impedance pattern can provide additional capabilitiesin conjunction with light-control. For example, it may be desirable totransport beads against a substrate-embedded impedance gradient toseparate beads on the basis of mobility.

Conduits connecting O and F need not be straight: as with tracksdirecting the motion of trains, conduits may be shaped in any desirablefashion (FIG. 1d ). A gated version of translocation (FIG. 1e ) permitsthe transport of particles from O to F only after the conduit is opened(or formed in real time) by a gating signal. This operation utilizes UVoxide patterning to establish two holding areas, O and F, and also lightcontrol to temporarily establish a conduit connecting O and F. Analternative implementation is based on an oxide embedded impedancegradient. A zone along the conduit is illuminated with sufficiently highintensity to keep out particles, thereby blocking the passage. Removal(or reduction in intensity) of the illumination opens the conduit. Inthe former case, light enables the transport of beads, while in thelatter case, light prevents the transport of beads.

The fundamental three-terminal operations are the merging and splittingof sets or arrays of beads (FIGS. 1f and 1g ). The merging of two arrays(FIG. 1f ) involves the previous two fundamental operations of“capture-and-hold”, applied to two spatially isolated sets of beads inlocations O1 and O2, and their respective channeling along mergingconduits into a common target area, and their eventual channeling,subsequent to mixing, or a chemical reaction, into the finaldestination, a third holding area, F. This is accomplished, under theconditions stated above, by invoking one-terminal and gated two-terminaloperations.

The splitting of an array into two subways (FIG. 1g ) is a special caseof a generally more complex sorting operation. Sorting involves theclassification of beads in a given set or array into one of two subsets,for example according to their fluorescence intensity. In the simplerspecial case, a given array, held in area O, is to be split into twosubarrays along a demarcation line, and subarrays are to be moved totarget areas F1 and F2. Under the conditions stated above, this isaccomplished by applying the “capture-and-hold” operation to the arrayin O. Conduits connect O to F1 and F2. High intensity illumination alonga narrowly focused line serves to divide the array in a defined fashion,again relying on gated translocation to control transport along conduitsaway from the holding area O. An even simpler version, leanedindiscriminate splitting, randomly assigns particles into F1 and F2 bygated translocation of the array in O into F1 and F2 after conduits areopened as described above.

FIGS. 4a and 4b show a variant in which beads in region O (FIG. 4a ) aresplit into multiple regions F1, F2, . . . Fn (FIG. 4b ). This reversiblesplitting of an aggregate or array into n subarrays, or clusters, isaccomplished, for carboxylated polystyrene spheres of 2 micron diameterat a concentration corresponding to an electrode coverage of a smallfraction of a monolayer, at a frequency of 500 Hz, by raising theapplied voltage from typically 5V (pp) to 20V (pp). This fragmentationof an array into smaller clusters reflects the effect of a field-inducedparticle polarization. The splitting is useful to distribute particlesin an array over a wider area of substrate for presentation to possibleanalytes in solution, and for subsequent scanning of the individualclusters with analytical instruments to make individual readings.

The three functional elements of the present invention described berthmay be also combined to yield additional fundamental operations tocontrol the orientation of anisotropic objects embedded in theelectroosmotic flow created by the applied electric field at theelectrode surface. The direction of the flow, in the plane of thesubstrate, is controlled by gradients in the impedance that are shapedin the manner described in connection with the channeling operation.This is used to controllably align anisotropic objects as illustrated inFIG. 1h , and may be applied to stretch out and align biomolecules, suchas DNA.

An additional fundamental operation that complements the previous set isthat of permanently anchoring an array to the substrate. This is bestaccomplished by invoking anchoring chemistries analogous to thoserelying on heterobifunctional cross-linking agents invoked to anchorproteins via amide bond formation. Molecular recognition, for examplebetween biotinylated particles and surface-anchored streptavidin,provides another class of coupling chemistries for permanent anchoring.

General Experimental Conditions

The functional elements, namely the electric-field induced assembly ofplanar particle arrays, the spatial modulation of the interfacialimpedance by means of UV-mediated oxide or surface-chemical patterningand finally, the control over the state of the interfacial impedance bylight which are used in the present invention, have been demonstrated inexperimental studies. These studies employed n-doped silicon wafers(resistivities in the range of 0.01 Ohm cm), capped with eitherthermally grown oxide layers of several thousand Angstrom thickness, orwith thin oxide layers, regrown after removal of the original “native”oxide in HF, under UV illumination from a deuterium source in thepresence of oxygen to typical thicknesses between 10 and 50 Angstroms.Lithographic patterning of thermally grown oxide employed standardprocedures implemented on a bench top (rather than a clean room) toproduce features in the range of several microns.

Surfaces were carefully cleaned in adherence with industry standard RCAand Piranha cleaning protocols. Substrates were stored in water producedby a Millipore cleaning system prior to use. Surfaces were characterizedby measuring the contact angle exhibited by a 20 microliter droplet ofwater placed on the surface and viewed (from the side) through atelescope. The contact angle is defined as the angle subtended by thesurface and the tangent to the droplet contour (in side view) at thepoint of contact with the surface. For example, a perfectlyhemispherical droplet shape would correspond to a contact angle of 90degrees. Surface chemical derivatization withmercapto-propyl-trimethoxysilane (2% in dry toluene) produced surfacesgiving typical contact angles of 70 degrees. Oxidation of the terminalthiol functionality under UV irradiation in the presence of oxygenreduced the contact angle to zero in less than 10 min of exposure to UVfrom the deuterium source. Other silane reagents were used in a similarmanner to produce hydrophobic surfaces, characterized by contact anglesin excess of 110 degrees.

Simple “sandwich” electrochemical cells were constructed by employingkapton film as a spacer between Si/SiOx and conductive indium tin oxide(ITO), deposited on a thin glass substrate. Contacts to platinum leadswere made with silver epoxy directly to the top of the ITO electrode andto the (oxide-stripped) backside of the Si electrode. In thistwo-electrode configuration, AC fields were produced by a functiongenerator, with applied voltages ranging up to 20 V and frequenciesvarying from DC to 1 MHz, high frequencies favoring the formation ofparticle chains connecting the electrodes. Currents were monitored witha potentiostat and displayed on an oscilloscope. For convenience,epi-fluorescence as well as reflection differential interferencecontrast microscopy employed laser illumination. Light-inducedmodulations in EIS impedance were also produced with a simple 100 Wmicroscope illuminator as well as with a 3 mW laser diode emitting lightat 650-680 nm.

Colloidal beads, both anionic and cationic as well as nominally neutral,with a diameter in the range from several hundred Angstroms to 20microns, stored in a NaN₂ solution, were employed.

Close attention was paid to colloidal stability to avoid non-specificinteractions between particles and between particles and the electrodesurface. Bacterial contamination of colloidal suspensions wasscrupulously avoided.

Typical operating conditions producing, unless otherwise indicated, mostof the results described herein, were: 0.2 mM NaN₂ (sodium azide)solutions, containing particles at a concentration so as to produce notmore than a complete monolayer of particles when deposited on theelectrode; applied DC potentials in the range of 1-4V, and AC potentialsin the range of 1-10V (peak-to-peak) and 500 Hz-10 kHz, with anelectrode gap of 50 microns; anionic (carboxylated polystyrene) beads of2 micron diameter, as well as (nominally neutral) polystyrene beads of2-20 micron diameter.

The method and apparatus of the present invention may be used in severaldifferent areas, examples of which are discussed in detail. Each exampleincludes background information followed by the application of thepresent invention to that particular application.

Example I Fabrication of Surfaces and Coatings with Designed Properties

The present invention may be used to fabricate planar surfaces andcoatings with designed properties. Specifically, the functional elementsof the present invention enable the formation of arrays composed ofparticles of a wide range of sizes (approximately 100 Angstrom to 10microns) and chemical composition or surface functionality in responseto AC or DC electric fields. These arrays may be placed and delineatedin designated areas of the substrate, and the interparticle spacing andinternal state of order within the array may be controlled by adjustingthe applied field prior to anchoring the array to the substrate. Thenewly formed surfaces display pre-designed mechanical, optical andchemical characteristics, and they may be subjected to furthermodification by subsequent treatment such as chemical cross-linking.

The mechanical and/or chemical modification of surfaces and coatingsprincipally determines the interaction between materials in a wide rangeof applications that depend on low adhesion (e.g., the familiar“non-stick” surfaces important in housewares) or low friction (e.g., toreduce wear in computer hard disks), hydrophobicity (the tendency torepel water, e.g., of certain fabrics), catalytic activity or specificchemical functionality to either suppress molecular interactions withsurfaces or to promote them. The latter area is of particular importanceto the development of reliable and durable biosensors and bioelectronicdevices. Finally, a large number of applications depend on surfaces ofdefined topography and/or chemical functionality to act as templatescontrolling the growth morphology of deposited materials or as “commandsurfaces” directing the alignment of optically active molecules indeposited thin organic films, as in liquid crystal display applications.

Extensive research has been devoted to the formation of surfaces byadsorption of thin organic films of known composition from the liquid orgas phase by several methods. Notwithstanding their seeming simplicityand wide-spread use, these methods can be difficult to handle inproducing reliable and reproducible results. In addition, molecularfilms are not well suited to produce surfaces displaying playing aregular topography.

An alternative approach to the problem is the modification of conductivesurfaces by electrophoretic deposition of suspended particulates. Thisis a widely used technique in industrial settings to produce paintcoatings of metal parts, and to deposit phosphor for display screens.The active deposition process significantly enhances the kinetics offormation (in contrast to passive adsorption of organic films fromsolution), an important consideration in practical applications.Electrophoretic deposition requires high DC electric fields and produceslayers in which particles are permanently adsorbed to the surface. Whileparticles in so-deposited monolayers are usually randomly distributed,the formation of polycrystalline monolayers of small (150 Angstrom) goldcolloids on carbon-coated copper grids is also known. However, the useof carbon-coated copper grids as substrates is not desirable in mostapplications.

Prior art methods have been described for the formation of orderedplanar arrays of particles under certain conditions. For example, theformation of ordered colloidal arrays in response to AC electric fieldson conductive indium tin oxide (ITO) electrodes is known. However, theresulting layers were composed of small patches of ordered arrays,randomly distributed over the surface of the otherwise bare ITOsubstrate. Arrays of monodisperse colloidal beads and globular proteinsalso have been previously fabricated by using convective flow andcapillary forces. However, this latter process has the disadvantage ofleaving deposited particle arrays immobilized and exposed to air, makingit difficult to modify arrays by subsequent liquid phase chemistry.

The present invention provides a method of forming planar arrays withprecise control over the mechanical, optical and chemical properties ofthe newly created layer. This method has several distinct advantagesover the prior art. These result from the combination of AC electricfield-induced array formation on insulating electrodes (Si/SiOx) thatare patterned by UV-mediated oxide regrowth. The process of the presentinvention enables the formation of ordered planar arrays from the liquidphase (in which particles are originally suspended) in designatedpositions, and in accordance with a given overall outline. Thiseliminates the above-stated disadvantages of the prior art, i.e., drystate; irregular or no topography, random placement within an aggregate,immobilization of particles and uncontrolled, random placement ofordered patches on the substrate.

An advantage of the present invention is that arrays are maintained bythe applied electric field in a liquid environment. The process leavesthe array in a state that may be readily disassembled, subjected tofurther chemical modification such as cross-linking, or made permanentby chemical anchoring to the substrate. Furthermore, the liquidenvironment is favorable to ensure the proper functioning of manyproteins and protein supramolecular assemblies of which arrays may becomposed. It also facilitates the subsequent liquid-phase deposition ofadditional layers of molecules (by chemical binding to beads or proteinsin the deposited layer), the cycling of arrays between states ofdifferent density and internal order (including complete disassembly ofthe array) in response to electric fields and the chemical cross-linkingof particles into two-dimensionally connected layers, or gels, formed,for example, of chemically functionalized silica spheres. The presentinvention can be practiced on insulating electrodes such as oxide-cappedsilicon, to minimize Faradaic processes that might adversely affectchemical reactions involved in the gelation process or in anchoring thearray to the substrate. The use of Si/SiOx electrodes also enables thecontrol of array placement by external illumination.

The formation of colloidal arrays composed of small particles inaccordance with the present invention provides a route to thefabrication of surfaces with relief structure on the scale of theparticle diameter. Aside from their optical properties, such“micro-rough” surfaces are of interest as substrates for the depositionof DNA in such a way as to alleviate steric constraints and thus tofacilitate enzyme access.

Particles to which the invention applies include silica spheres, polymercolloids, lipid vesicles (and related assemblies) containing membraneproteins such as bacteriorhodopsin (bR)⁻ a light-driven proton pump thatcan be extracted in the form of membrane patches and disks or vesicles.Structured and functionalized surfaces composed of photoactive pigmentsare of interest in the context of providing elements of planar opticaldevices for the development of innovative display and memory technology.Other areas of potential impact of topographically structured andchemically functionalized surfaces are the fabrication of templatesurfaces for the controlled nucleation of deposited layer growth andcommand surfaces for liquid crystal alignment. The present inventionalso enables the fabrication of randomly heterogeneous compositesurfaces. For example, the formation of arrays composed of a mixture ofhydrophobic and hydrophilic beads of the same size creates a surfacewhose wetting and lubrication characteristics may be controlled by thecomposition of the deposited mixed head array. In this way, the locationof the individual-beads is random, but the relative proportion of eachtype of bead within the allay is controllable.

Example II Assembly of Lens Arrays and Optical Diffraction Elements

The present invention can be used to fabricate lens arrays and othersurface-mounted optical elements such as diffraction gratings. Thefunctional elements of the present invention enable the placement anddelineation of these elements on ITO, facilitating integration withexisting planar display technology, and on Si/SiOx, facilitatingintegration with existing silicon-based device technology.

Silica or other oxide particles, polymer latex beads or other objects ofhigh refractive index suspended in an aqueous solution, will refractlight. Ordered planar arrays of beads also diffract visible light,generating a characteristic diffraction pattern of sharp spots. Thiseffect forms the basis of holographic techniques in optical informationprocessing applications.

A.—The present invention provides for the use of arrays of refractivecolloidal beads as light collection elements in planar array formats inconjunction with low light level detection and CCD imaging. CCD andrelated area detection schemes will benefit from the enhanced lightcollection efficiency in solid-phase fluorescence or luminescencebinding assays.

This assay format relies on the detection of a fluorescence signalindicating the binding of probes to bead-anchored targets in thevicinity of the detector. To maximize through-put, it is desirable tomonitor simultaneously as many binding events as possible. It is herethat any formation by the methods of the present invention isparticularly valuable because it facilitates the placement and tightpacking of beads in the target area monitored by the CCD detector, whilesimultaneously providing for the additional benefit of lensing actionand the resulting increase in light collection efficiency.

Increased collection efficiency has been demonstrated in experimentsemploying individual, large (10 micron diameter) polystyrene beads aslensing elements to image small (1 micron diameter) fluorescentpolystyrene beads. Under the experimental conditions set forth dove anapplied voltage of 5V (pp) at 300 Hz induced the collection of smallparticles under individual large beads within a second. This is shown inFIG. 5, where small beads alone, e.g., 52, appear dim, whereas smallbeads, e.g., 54, gathered under a large bead 56 appear brighter andmagnified. The small beads redisperse when the voltage is turned off.

B.—The use of colloidal bead arrays as diffraction gratings and thus asholographic elements is brown. Diffraction gratings have the property ofdiffracting light over a narrow range of wavelengths so that for givenangle of incidence and wavelength of the illuminating light, the arraywill pass only a specific wavelength (or a narrow band of wavelengthscentered on the nominal value) that is determined by the inter-particlespacing. Widely discussed applications of diffraction gratings rangefrom simple wavelength filtering to the more demanding realization ofspatial filters and related holographic elements that are essential inoptical information processing.

The present invention provides for a rapid and well controlled processof forming planar arrays in a state of crystalline order which willfunction as surface-mounted optical diffraction elements. In addition,the resulting surfaces may be designed to display topographical reliefto enhance wave-length selective reflectivity. These arrays may beformed in designated areas on a substrate surface. In contrast to theslow and cumbersome prior art method of fabricating such arrays by wayof forming equilibrium crystals in aqueous solutions of low saltcontent, the present invention provides a novel approach to rapidly andreliably fabricate particle arrays at a solid-liquid interface. Thisapproach relies on field-induced formation of arrays to bigger theprocess, and on UV-mediated patterning or light control to position andshape the arrays. In addition, the inter-particle distance, and internalstate of order and hence the diffraction characteristics of the array,may be fine-tuned by adjusting the applied electric field. For example,a field-induced, reversible order-disorder transition in the array willalter the diffraction pattern from one composed of sharp spots to onecomposed of a diffuse ring. The assembly of such arrays on the surfaceof silicon wafers, as described herein, provides a direct method ofintegration into existing microelectronic designs. Arrays may be lockedin place by chemical coupling to the substrate surface, or by relying onvan der Waals attraction between beads and substrate.

Example III A Novel Mechanism for the Realization of a Particle-BasedDisplay

The present invention provides the elements to implement lateralparticle motion as a novel approach to the realization of aparticle-based display. The elements of the present invention providefor the control of the lateral motion of small particles in the presenceof a pre-formed lens array composed of large, refractive particles.

Colloidal particulates have been previously employed in flat-paneldisplay technology. The operating principle of these designs is based onelectrophoretic motion of pigments in a colored fluid confined betweentwo planar electrodes. In the OFF (dark) state, pigments are suspendedin the fluid, and the color of the fluid defines the appearance of thedisplay in that state. To attain the ON (bright) state, particles areassembled near the front (transparent) electrode under the action of anelectric field. In this latter state, incident light is reflected by thelayer of particles assembled near the electrode, and the display appearsbright. Prototype displays employing small reflective particles inaccordance with this design are known. However, these displays sufferedfrom a number of serious problems including: electrochemical degradationand lack of colloidal stability as a result of prolonged exposure to thehigh DC electric fields required to achieve acceptable switching speeds;and non-uniformities introduced by particle migration in response tofield gradients inherent in the design of the addressing scheme.

The present invention provides a novel mechanism for the design of aparticle-based display which takes advantage of electric field-inducedarray formation as well as controlled, field-induced lateral particledisplacements. First, a lens array composed of colloidal beads isformed. This lens array also serves as a spacer array to maintain awell-defined gap between the bottom electrode and the top electrode thatmay now be placed over the (pre-formed) array. This facilitatesfabrication of uniform flat panel displays with a narrow gap that isdetermined by the particle diameter.

Next, small colloidal particles are added to the electrolyte solution inthe gap. These may be fluorescent, or may be reflecting incident whitelight. Under the action of an AC electric field of appropriatefrequency, these small particles can be moved laterally to assemblepreferentially within the footprint of a larger bead. When viewedthrough a larger bead, small fluorescent beads assembled under a largebead appear bright as a result of the increased light collectionefficiency provided by the leasing action of the huge bead; this is theON state (FIG. 5). When moved outside the footprint of the larger bead,particles appear dim, and may be made entirely invisible by appropriatemasking; this is the OFF state. The requisite lateral particle motionmay be induced by a change in the applied voltage or a change in lightintensity. Each large or leasing bead introduces a lateral nonuniformityin the current distribution within the electrolyte because the currentis perturbed by the presence of each lensing bead.

In contrast to the prior art displays, the present invention employs AC,not DC fields, and insulating (rather than conductive) electrodes,thereby minimizing electrochemical degradation. The lateralnon-uniformity introduced by the lens array is desirable because itintroduces lateral gradients in the current distribution within thedisplay cell. These gradients mediate the lateral motion of small beadsover short characteristic distances set by the diameter of the largelensing beads, to effect a switching between ON and OFF states. Thus,the present invention readily accommodates existing technology foractive matrix addressing.

Example IV Layout-Preserving Transfer of Bead Suspensions fromMicrotiter Plate to Planar Cell

The present invention provides a method to transfer suspensions of headsor biomolecules to the electrode surface in such a way as to preservethe spatial encoding in the original arrangement of reservoirs, mostcommonly the conventional 8×12 arrangement of wells in a microtiterplate. Such a fluid transfer scheme is of significant practicalimportance given that compound libraries are commonly handled andshipped in 8×12 wells.

The present invention utilizes chemical patterning to define individualcompartments for each of M×N sets of beads and confine them accordingly.In the present instance, patterning is achieved by UV-mediatedphotochemical oxidation of a monolayer of thiol terminated alkylsilanethat is chemisorbed to the Si/SiOx substrate. Partial oxidation of thiolmoities produces sulfonate moities and renders the exposed surfacecharged and hydrophilic. The hydrophilic portions of the surface, in theform of a grid of squares or circles, will serve as holding areas.

In accordance with the present invention, the first function ofsurface-chemical patterning into hydrophilic sections surrounded byhydrophobic portions is to ensure that droplets, dispensed fromdifferent wells, will not fuse once they are in contact with thesubstrate. Consequently, respective bead suspensions will remainspatially isolated and preserve the lay-out of the original M×N wellplate. The second role of the surface chemical patterning of the presentinvention is to impose a surface charge distribution, in the form of theM×N grid pattern, which ensures that individual bead arrays will remainconfined to their respective holding areas even as the liquid phasebecomes contiguous.

The transfer procedure involves the steps illustrated in FIGS. 6a-c .First, as shown in sideview in FIG. 6a , the M×N plate of wells 62 isregistered with the pattern 64 on the planar substrate surface. Wellbottoms 62 are pierced to allow for the formation of pendant drops ofsuspension or, preferably, the process is facilitated by a fixture (notshown) providing M×N effective funnels to match the geometric dimensionsof the M×N plate on the top and reduce the size of the dispensing end.Such a dispensing fixture will also ensure the precise control ofdroplet volumes, adjusted so as to slightly overfill the target holdingarea on the patterned substrate surface. The set of M×N drops is thendeposited by bringing them in contact with the hydrophilic holding areasof the pre-patterned substrate and relying on capillary action.

Next, the plate is retracted, and the top electrode is carefully loweredto form the electrochemical cell, first making contact as shown in FIG.6b , with individual liquid-filled holding areas on the substrate towhich suspensions are confined. Overfilling ensures that contact is madewith individual suspensions. The electric field is now turned on toinduce array formation in the M×N holding areas and to ensure thepreservation of the overall configuration of the M×N sets of beads whilethe gap is closed further (or filled with additional buffer) toeventually fuse the individual droplets of suspension into a contiguousliquid phase as shown in FIG. 6c . In the fully assembled cell of FIG.6c , while the droplets are fused together, the beads from each dropletare maintained in and isolated in their respective positions, reflectingthe original M×N arrangement of wells. The present invention thusprovides for the operations required in this implementation of alayout-preserving transfer procedure to load planar electrochemicalcells.

Example V Preparation of Heterogeneous Panels of Particles

The present invention provides a method to produce a heterogeneous panelof beads and potentially of biochemicals for presentation to analytes inan adjacent liquid. A heterogeneous panel contains particles orbiomolecules which differ in the nature of the chemical or biochemicalbinding sites they offer to analytes in solution. In the event ofbinding, the analyte is identified by the coordinates of the bead, orcluster of beads, scoring positive. The present method relies on thefunctional elements of the invention to assemble a planar array of amulti-component mixture of beads which carry chemical labels in the formof tag molecules and may be so identified subsequent to performing theassay.

Diagnostic assays are frequently implemented in a planar format of aheterogeneous panel, composed of simple ligands, proteins and otherbiomolecular targets. For example, in a diagnostic test kit, aheterogeneous panel facilitates the rapid testing of a given analyte,added in solution, against an entire set of targets. Heterogeneouspanels or proteins are of great current interest in connection with theemerging field of proteome research. The objective of this research isto identify, by scanning the panel with sensitive analytical techniquessuch as mass spectrometry, each protein in a multi-component mixtureextracted from a cell and separated by two-dimensional gelelectrophoresis. Ideally, the location of each spot uniquely correspondsto one particular protein. This analysis would permit, for example, thedirect monitoring of gene expression levels in a cell during aparticular point in its cycle or at a given stage during embryonicdevelopment.

The fabrication of an array of heterogeneous targets is central torecently proposed strategies of drug screening and DNA mutation analysisin a planar format. The placement of ligands in a specific configurationon the surface of a planar substrate serves to maintain a key to theidentity of any one in a large set of targets presented simultaneouslyto an analyte in solution for binding or hybridization. In an assayrelying on fluorescence, binding to a specific target will create brightspots on the substrate whose spatial coordinates directly indicate theidentity of the target.

Three principal strategies have been previously employed to fabricateheterogeneous panels. First, protein panels may be created bytwo-dimensional gel electrophoresis, relying on a DC electric field toseparate proteins first by charge and then by size (or molecularweight). Even after many years of refinement, this technique yieldsresults of poor reproducibility which are generally attributed to thepoorly defined properties of the gel matrix.

Second, individual droplets, drawn from a set of reservoirs containingsolutions of the different targets, may be dispensed either by hand orby employing one of several methods of automated dispensing (or“printing”; see e.g., Schena et al., Science 270, 467-470 (1995), thecontents of which are incorporated herein by reference). Printing hasbeen applied to create panels of oligonucleotides intended for screeningassays based on hybridization. Printing leaves a dried sample and maythus not be suitable for proteins that would denature under suchconditions. In addition, the attendant fluid handling problems, inherentin maintaining, and drawing samples from a large number of reservoirsare formidable.

Third, target ligands may be created by invoking a variant of solidphase synthesis based on a combinatorial strategy of photochemicallyactivated elongation reactions. This approach has been limited by veryformidable technical problems in the chemical synthesis of even thesimplest, linear oligomers. The synthesis of non-linear compounds inthis planar geometry is extremely difficult.

The present invention of forming heterogeneous panels requires thechemical attachment of target ligands to beads. Ligands may be coupledto beads “off-line” by a variety of well established coupling reactions.For present purposes, the bead identity must be chemically encoded so itmay be determined as needed. Several methods of encoding, or binaryencoding, of beads are available. For example, short oligonucleotidesmay serve the purpose of identifying a bead via their sequence which maybe determined by microscale sequencing techniques. Alternatively,chemically inert molecular tags may be employed that are readilyidentified by standard analytical techniques.

In contrast to all prior art methods, the present invention provides anovel method to create heterogeneous panels by in-situ, reversibleformation of a planar array of “encoded” beads in solution adjacent toan electrode. The array may be random with suspect to chemical identitybut is ordered with respect to spatial position. This procedure offersseveral advantages. First, it is reversible so that the panel may bedisassembled following the binding assay to discard beads scoringnegative. Positive beads may be subjected to additional analysis withoutthe need for intermediate steps of sample retrieval, purification ortransfer between containers. Second, the panel is foraged when needed,that is, either prior to performing the actual binding assay, orsubsequent to performing the assay on the surface of individual beads insuspension. The latter mode minimizes potential adverse effects that canarise when probes bind to planar target surfaces with a highconcentration of target sites. Third, to accommodate scanning probeanalysis of individual beads, interparticle distances within the arraymay be adjusted by field-induced polarization or by the addition ofinert spacer particles that differ in size front the encoded beads. FIG.7 shows the use of small spacer beads 72 for separating encoded beads74. As shown, the spacing of beads 74 is greater than the spacing ofcomparable beads in FIG. 4b . Finally, UV-mediated oxide regrowth, asprovided by the present invention, readily facilitates the embedding ofa grid pattern of selected dimension into the substrate to ensure theformation of small, layout-preserving subarrays in the low-impedancefields of the grid.

To create the panel, a multi-component mixture of beads carrying, forexample, compounds produced by bead-based combinatorial chemistry, isplaced between electrodes. Each type of bead may be present in multiplecopies. Arrays are formed in response to an external field in adesignated area of the electrode surface. This novel approach of in-situassembly of panels relies on beads that carry a unique chemical label,or code, to permit their identification subsequent to the completion ofa binding assay. This invention facilitates on-line tagging of bouts byway of a photochemical bead-coloring method. Selected beads in an arrayare individually illuminated by a focused light source to trigger acoloring reaction on the bead surface or in the bead interior toindicate a positive assay score. Beads so marked can be subsequentlyseparated from unmarked beads by a light-activated sorting methoddescribed herein. Numerous UV-activated reactions are available toimplement this bead-coloring method.

The present invention provides for several methods of discarding beadswith negative scores, typically the vast majority, while retaining thosewith positive scores. This method take advantage of the fact that, incontrast to all prior it methods, the array represents a temporaryconfiguration of particles that is maintained by the applied electricfield and may be rearranged or disassembled at will. This capability,along with the fact that biomolecules are never exposed to air (as inthe prior art method of printing) facilitates the in-situ concatenationof analytical procedures that require the heterogeneous panel inconjunction with subsequent, “downstream” analysis.

First, if positive beads are clustered in a subsection of the array, thelight-controlled array splitting operation of the present invention maybe invoked to dissect the array so as to discard negative portions ofthe array (or recycle them for subsequent use). Second, if positive andnegative beads are randomly interspersed, a fluorescence-activatedsorting method, implemented on the basis of the present invention in aplanar format, as described herein, may be invoked. In the case offluorescence-activated sorting, positive and negative beads may beidentified as bright and dark objects, respectively. In the special casethat only a few positive beads stand out, these may be removed from thearray by locking onto them with optical tweezers, a tool to trap and/ormanipulate individual refractive particles under illumination, anddisassembling the array by removing the field, or subjecting the entirearray to lateral displacement by the fundamental operations of thepresent invention.

The typical task in screening a large set of compounds is one of Lookingfor a very small number of positive events in a vast number of tests.The set of discarded beads will typically involve the majority at eachstage in the assay. The procedure of the present invention thereforeminimizes the effort invested in negative events, such as thechallenging in-situ synthesis of target ligands irrespective of whetheror not they will prove to be of interest by binding a probe offered insolution.

The method of forming a heterogeneous panel according to the presentinvention contains beads of each type in generally random assembly. Thecreation of a heterogeneous panel with each position in the panelcontaining a cluster of beads of the same type, that is, beadsoriginating in the same reservoir (FIG. 6a ), may be desirable so as toensure a sufficiently large number of positive events to facilitatedetection. A practical solution follows from the application of thelayout-preserving fluidic transfer scheme described herein. In thisprocedure, beads from an M×N well plate are transferredlayout-preservingly onto a chemically patterned substrate in such a wayas to preserve the spatial encoding of bead identities.

Example VI Binding and Functional Assays in Planar Bead Array Format

The present invention can be used to implement mixed-phase bindingassays as well as certain functional assays in a planar array format.Several combinations are possible reflecting the presence of probe ortarget in solution, on the surface of colloidal beads, or on theelectrode surface. The methods of the present invention facilitate theformation of a planar array to present targets to probes in solutionprior to performing the binding assay (“preformed” array; FIG. 8).Alternatively, a planar array of beads may be formed in front of adetector surface subsequent to performing the binding assay insuspension (“postformed” array; FIG. 8). The present invention alsoprovides the methods to implement functional assays by enabling theassembly of certain cell types adjacent to a planar detector or sensorsurface to monitor the effects of exposure of the cells to smallmolecule drugs in solution.

Binding assays, particularly those involving proteins such as enzymesand antibodies, represent a principal tool of medical diagnostics. Theyare based on the specific biochemical interaction between a probe, suchas a small molecule, and a target, such as a protein. Assays facilitatethe rapid detection of small quantities of an analyte in solution withhigh molecular specificity. Many procedures have been designed toproduce signals to indicate binding, either yielding a qualitativeanswer (binding or no binding) or quantitative results in the form ofbinding or association constants. For example, when an enzyme binds ananalyte, the resulting catalytic reaction may be used to generate asimple color change to indicate binding, or it may be coupled to otherprocesses to produce chemical or electrical signals from which bindingconstants are determined. Monoclonal antibodies, raised from a singlecommon precursor, may be prepared to recognize virtually any giventarget, and immunoassays, based on antibody-antigen recognition andbinding, have developed into an important diagnostic tool. As withenzyme binding, antibody binding of an antigenic analyte may be detectedby a variety of techniques including the classic method of enzyme-linkedimmunoassays (ELISA) in which the reaction of an antibody-coupled enzymeis exploited as an indicator. A common and conceptually simple schemeensures the detection of antibody binding to a target analyte bysupplying a fluorescently labeled second antibody that recognizes thefirst (or primary) antibody.

Binding assays involving soluble globular proteins are often performedin solution to ensure unbiased interactions between protein and target.Such liquid phase assays, especially when performed at lowconcentrations of target or probe, minimize potential difficulties thatmay arise when either target or probe are present in abundance or inclose proximity. By the same token, the kinetics tend to be slow.Cooperative effects, such as crowding, arising from the close proximityof probes must be carefully controlled when either probe or target ischemically anchored to a solid substrate.

Nonetheless, this latter solid phase format of binding assays is alsovery commonly employed whenever the situation demands it. For example,the presence of a protein on the surface of a cell may be exploited in“panning” for the cells that express this protein in the presence ofmany other cells in a culture that do not: desired cells attachthemselves to the surface of a container that is pre-coated with a layerof a secondary antibody directed against a primary antibody decoratingthe desired cell-surface protein. Similarly, certain phages may begenetically manipulated to display proteins on their surface, and thesemay be identified by a binding assay involving a small molecule probesuch as an antigen if the protein displayed is an antibody (Watson etal., “Recombinant DNA”, 2nd Edition (Scientific American Book, Freemanand Co., New York, N.Y., 1983), the contents of which are incorporatedherein by reference). In addition, the planar geometry accommodates avariety of optical and electrical detection schemes implemented intransducers and sensors.

A combination of liquid phase and solid phase assay may be developed byusing beads that are decorated with either probe or target, as inprocedures that employ decorated magnetic beads for sample preparationor purification by isolating binding from non-binding molecules in agiven multi-component mixture. Recent examples of the use of these beadsinclude the purification of templates for DNA sequencing applications orthe extraction of mRNAs from (lysed) cells by hybridization to beadsthat are decorated with poly-adenine (polyA) residues.

Functional assays involving suitable types of cells are employed tomonitor extracellular effects of small molecule drugs on cellmetabolism. Cells are placed in the immediate vicinity of a planarsensor to maximize the local concentration of agents released by thecell or to monitor the pH.

The present invention provides the means to implement mixed phasebinding assays in a planar geometry with a degree of flexibility andcontrol that is not available by prior art methods. Thus, it offers theflexibility of forming, in-situ, reversibly and under external spatialcontrol, either a planar panel of target sites for binding of analytepresent in an adjacent liquid phase, or a planar array of probe-targetcomplexes subsequent to performing a binding assay in solution. Bindingmay take place at the surface of individual beads suspended in solution,at the surface of beads pre-assembled into arrays adjacent to theelectrode surface, or at the electrode surface itself. Either the targetor probe molecule must be located on a bead to allow for a bead-basedassay according to the present invention. As shown in FIG. 8, if theprobe molecule P is located on a bead, then the target molecule T may beeither in solution, on a bead or on the electrode surface. The converseis also true.

For example, the methods of the present invention may be used toimplement panning, practiced to clone cell surface receptors, in a farmore expeditious and controlled manner than is possible by the prior artmethod. Given a substrate that has been coated with a layer of antibodydirected against the sought-after cell surface protein, the presentinvention facilitates the rapid assembly of a planar array of cells ordecorated beads in proximity to the layer of antibodies and thesubsequent disassembly of the away to leave behind only those cells orbeads capable of forming a complex with the surface-bound antibody.

A further example of interest in this category pertains to phagedisplays. This technique may be employed to present a layer of proteintargets to bead-anchored probes. Bead arrays may now be employed toidentify a protein of interest. That is, beads are decorated with smallmolecule probes and an array is formed adjacent to the phage display.Binding will result in a probe-target complex that retains beads whileothers are removed when the electric field is turned off, or whenlight-control is applied to remove beads from the phage display. Ifbeads are encoded, many binding tests may be carried out in parallelbecause retained beads may be individually identified subsequent tobinding.

The methods of the present invention readily facilitate competitivebinding assays. For example, subsequent to binding of a fluorescentprobe to a target-decorated bead in solution and the formation of aplanar bead array adjacent to the electrode, fluorescent areas withinthe array indicate the position of positive targets, and these may befurther probed by subjecting them to competitive binding. That is, whilemonitoring the fluorescence of a selected section of the planar array,an inhibitor (for enzyme assays) or other antagonist (of known bindingconstant) is added to the electrochemical cell, and the decrease influorescence originating from the region of interest is measured as afunction of antagonist concentration to determine a binding constant forthe original probe. This is an example of a concatenation of analyticalsteps that is enabled by the methods of the present invention.

The fact that a probe-target complex is fixed to a colloidal bead, as inthe methods of the present invention, conveys practical advantagesbecause this facilitates separation of positive from negative events.Particularly when solid phase assays are performed on a planarsubstrate, an additional advantage of planar bead arrays is theenhancement of light collection efficiency provided by the beads, asdiscussed herein.

If desired, beads may serve strictly as delivery vehicles for smallmolecule probes. That is, an array of probe-decorated beads is formedadjacent to a target-decorated surface in accordance with the methods ofthe present invention UV-activated cleavage of the probe from the beadsupport will ensure that the probe is released in close proximity to thetarget layer, thereby enhancing speed and efficiency of the assay. Theidentity of the particular probe interacting with the target may beascertained from the positional location of the bead delivering theprobe.

The methods of the present invention apply not only to colloidal beadsof a wide variety (bat need no special preparative procedures to makethere magnetic, for example), but also to lipid vesicles and cells thatare decorated with, or contain embedded in their outer wall, eitherprobe or target. The methods of the present invention may therefore beapplied not only to bead-anchored soluble proteins but potentially tointegral membrane receptors or to cell surface receptors.

In particular, the rapid assembly of cells in a designated area of thesubstrate surface facilitates the implementation of highly parallelcell-based functional assays. The present invention makes it possible toexpose cells to small molecule drug candidates in solution and rapidlyassemble them in the vicinity of a sensor embedded in the electrodesurface, or to expose pre-assembled cells to such agents that arereleased into the adjacent liquid phase. In the simplest case, all cellswill be of the same type, and agents will be administered sequentially.Even in this sequential version, electrokinetic mixing will enhancethrough-put. However, as described herein, the methods of the presentinvention also enable the parallel version of binding assays and thus offunctional assays in a planar format by encoding the identity ofdifferent cells by a “Layout-Preserving Transfer” process from an 8×12well plate, as discussed herein, and to isolate cells scoring positiveby providing feed-back from a spatially resolved imaging or sensingprocess to target a specific location in the array of cells.

Example VII Separation and Sorting of Beads and Particles

The present invention can be used to implement several procedures forthe separation and sorting of colloidal particles and biomolecules in aplanar geometry. Specifically, these include techniques of lateralseparation of beads in mixtures. Individual beads may be removed from anarray formed in response to an electric field by the application ofoptical tweezers.

The separation of components in a given mixture of chemical compounds isa fundamental task of analytical chemistry. Similarly, biochemicalanalysis frequently calls for the separation of biomolecules, beads orcells according to size and/or surface charge by electrophoretictechniques, while the sorting (most commonly into just two sub-classes)of suspended cells or whole chromosomes according to optical propertiessuch as fluorescence emission is usually performed using field-flowfractionation including flow cytometry and fluorescence-activated cellsorting.

In a planar geometry, bead mixtures undergoing diffusion have beenpreviously separated according to mobility by application of an ACelectric field in conjunction with lithographic patterning of theelectrode surface designed to promote directional drift. Essentially,the AC or pulsing electric field is used to move small beads in aparticular direction over a period of time. Capillary electrophoresishas been implemented in a planar geometry, see e.g., B. B. Haab and R.A. Mathies, Anal. Chem. 67, 3253-3260 (1995), the contents of which areincorporated herein by reference.

The methods of the present invention may be applied in several ways toimplement the task of separation, sorting or isolation in a planargeometry. In contrast to the prior art approaches, the present inventionprovides a significant degree of flexibility in selecting from amongseveral available procedures, the one best suited to the particular taskat hand. In some cases, more than one separation technique may beapplied, and this provides the basis for the implementation oftwo-dimensional separation. That is, beads may be separated according totwo different physical-chemical characteristics. For example, beads mayfirst be separated by size and subsequently, by raising the appliedfrequency to induce chain formation, by polarizability. This flexibilityoffers particular advantages in the context of integrating analyticalfunctionalities in a planar geometry. Several techniques will now bedescribed.

i) The present invention may be used to implement “sieving” in lateral,electric field-induced flow on surfaces patterned by UV-mediated oxideregrowth to sort beads in a mixture by size. The fundamental operationsof the invention are invoked to set up directed lateral particle motionalong conduits laid out by UV-mediated oxide regrowth. Conduits aredesigned to contain successively narrower constrictions through whichparticles must pass. Successively finer stages allow only successivelysmaller particles to pass in this “sieving” mechanism (FIG. 9a ). Asshown in FIG. 9a , the primary particle flow is in the direction left toright, while a transverse flow is established in the top to bottomdirection utilizing an oxide profile as shown. Additionally, rows ofbarriers 92 made from thick oxide are positioned along the conduit withthe spacing between the barriers in each row decreasing in thetransverse direction. As the particles move along the conduit, the rowsof barriers act to separate out smaller particles in the transversedirection. In contrast to previous methods based on electrophoreticseparation, large DC electric fields, and the attendant potentialproblem of electrolysis and interference from electroosmotic flow in adirection opposite to the field-directed particle transport, the presentinvention uses AC electric fields and lateral gradients in interfacialimpedance to produce transport. The present method has the advantage ofavoiding electrolysis and it takes explicit advantage of electroosmoticflow to produce and control particle transport.

In addition, the use of Si/SiOx electrodes enables the use of thelight-control component of the present invention to modify lateraltransport of beads in real time. For example, external illumination maybe employed to locally neutralize the lateral impedance gradient inducedby UV-mediated oxide regrowth. Particles in these neutral “zones” wouldno longer experience any net force and come to rest. This principle maybe used as a basis for the implementation of a scheme to locallyconcentrate particles into sharp bands and thereby to improve resolutionin subsequent separation.

ii) The present invention may be used to implement “zone refining”, aprocess of excluding minority components of a mixture by size or shapefrom a growing crystalline array of majority component. This processexplicitly depends on the capabilities of the present invention toinduce directional crystallization.

The process of zone refining is employed with great success in producinglarge single crystals of silicon of very high purity by excludingimpurities from the host lattice. The concept is familiar from thestandard chemical procedure of purification by recrystallization inwhich atoms or molecules that are sufficiently different in size, shapeor charge from the host species so as not to fit into the forming hostcrystal lattice as a substitutional impurity, are ejected into solution.

By enabling the growth of planar arrays, in a given direction and at acontrolled rate, the present invention facilitates the implementation ofan analogous zone refining process for planar arrays. The most basicgeometry is the linear geometry. A multi-component mixture of beads ofdifferent sizes and/or shapes is first captured in a rectangular holdingarea on the surface, laid out by UV-patterning. Next, crystallization isinitiated at one end of the holding area by illumination and allowed toslowly advance across the entire holding area in response to anadvancing pattern of illumination. In general, differences ofapproximately 10% in bead radius trigger ejection.

iii) The present invention may be used to implement fractionation in atransverse flow in a manner that separates particles according tomobility.

Field-flow fractionation refers to an entire class of techniques thatare in wide use for the separation of molecules or suspended particles.The principle is to separate particles subjected to fluid flow in afield acting transverse to the flow. A category of such techniques issubsumed under the heading of electric-field flow fractionation of whichfree-flow electrophoresis is a pertinent example because it iscompatible with a planar geometry. Free-flow electrophoresis employs thecontinuous flow of a replenished buffer between two narrowly spacedplates in the presence of a DC electric field that is applied in theplane of the bounding plates transverse to the direction of fluid flow.As they traverse the electric field, charged particles are deflected inproportion to their electrophoretic mobility and collected in separateoutlets for subsequent analysis. In contrast to conventionalelectrophoresis, free-flow electrophoresis is a continuous process withhigh throughput and it requires no supporting medium such as a gel.

The present invention enables the implementation of field-flowfractionation in a planar geometry. As previously discussed herein,impedance gradients imposed by UV-oxide profiling serve to mediateparticle motion along the electrode surface in response to the externalelectric field. In a cell with a narrow gap, the resultingelectrokinetic flow has a “plug” profile and this has the advantage ofexposing all particles to identical values of the flow velocity field,thereby minimizing band distortions introduced by the parabolic velocityprofile of the laminar flow typically employed in free-flowelectrophoresis.

A second flow field, transverse to the primary flow direction, may beemployed to mediate particle separation. This deflecting flow may begenerated in response to a second impedance gradient. A convenientmethod of imposing this second gradient is to take advantage of UV-oxidepatterning to design appropriate flow fields. Both longitudinal andtransverse flow would be recirculating and thus permit continuousoperation even in a closed cell, in contrast to any related prior arttechnique.

Additional flexibility is afforded by invoking the light-controlcomponent of the present invention to illuminate the substrate with astationary pattern whose intensity profile in the direction transverseto the primary fluid flow is designed to induce the desired impedancegradient and hence produce a transverse fluid flow. (FIG. 9b ). This hasthe significant advantage of permitting selective activation of thetransverse flow in response to the detection of a fluorescent beadcrossing a monitoring window upstream. Non-fluorescent beads would notactivate the transverse flow and would not be deflected. This procedurerepresents a planar analog of flow cytometry, or fluorescence-activatedcell sorting.

iv) The invention may be used to induce the formation of particle chainsin the direction normal to the plane of the electrode. The chainsrepresent conduits for current transport between the electrodes andtheir formation may reflect a field-induced polarization. Chains aremuch less mobile in transverse flow than are individual particles sothat this effect may be used to separate particles according to thesurface properties that contribute to the net polarization. The effectof reversible chain formation has been demonstrated under theexperimental conditions stated herein. For example, the reversibleformation of chains occurs, for carboxylated polystyrene beads of 1micron diameter, at a voltage of 15 V (pp) at frequencies in excess of 1MHz.

v) The invention may be used to isolate individual beads from a planararray.

Fluorescence binding assays in a planar array format, as describedherein, may produce singular, bright beads within a large array,indicating particularly strong binding. To isolate and retrieve thecorresponding beads, optical tweezers in the form of a sharply focusedlaser spot, may be employed to lock onto an individual bead of interest.The light-control component of the present invention may be used inconjunction with the optical tweezers to retrieve such an individualbead by moving the array relative to the bead, or vice versa, or bydisassembling the array and retaining only the marked bead. This is arather unique capability that will be particularly useful in the contextof isolating beads in certain binding assays.

Commercial instrumentation is available to position optical tweezers inthe field of a microscope. Larger scale motion is facilitated bytranslocating the array in-situ or simply by moving the external samplefixture. This process lends itself to automation in conjunction with theuse of peak-finding image analysis software and feedback control.

vi) The invention may be used to implement a light-induced arraysectioning (“shearing”) operation to separate fluorescent, or otherwisedelineated portions of an array from the remainder. This operation makesit possible to segment a given array and to isolate the correspondingbeads for downstream analysis.

The basis for the implementation of this array segmentation is thelight-control component of the present invention, in the mode of drivingparticles from an area of a Si/SiOx interface that is illuminated withhigh intensity. It is emphasized here that this effect is completelyunrelated to the light-induced force on beads that underlies the actionof optical tweezers. The present effect which operates on large sets ofparticles, was demonstrated under the experimental conditions statedherein using a 100 W illuminator on a Zeiss UEM microscope operated inepi-illumination. A simple implementation is to superimpose, on theuniform illumination pattern applied to the entire array, a line-focusedbeam that is positioned by manipulation of beam steering elementsexternal to the microscope. Beads are driven out of the illuminatedlinear portion. Other implementations take advantage of two separatelycontrolled beams that are partially superimposed. The linear sectioningcan be repeated in different relative orientations of shear and array.

Example VIII Screening for Drug Discovery in Planar Geometry

The functional elements of the present invention may be combined toimplement procedures for handling and screening of compound andcombinatorial libraries in a planar format. The principal requisiteelements of this task are: sample and reagent delivery from the set oforiginal sample reservoirs, commonly in a format of 8×12 wells in amicroliter plate, into a planar cell; fabrication of planar arrays oftargets or of probe-target complexes adjacent to the planar electrodesurface prior to or subsequent to performing a binding assay; evaluationof the binding assay by imaging the spatial distribution of markerfluorescence or radioactivity, optionally followed by quantitativepharmacokinetic measurements of affinity or binding constants; isolationof beads scoring positive, and removal from further processing of otherbeads; and collection of specific beads for additional downstreamanalysis. The present invention relates to all of these elements, andthe fundamental operations of the invention provide the means toconcatenate these procedures in a planar format.

A central issue in the implementation of cost-effective strategies formodern therapeutic drug discovery is the design and implementation ofscreening assays in a manner facilitating high throughput whileproviding pharmacokinetic data as a basis to select promising drug leadsfrom a typically vast library of compounds. That is, molecularspecificity for the target, characterized by a binding constant, is animportant factor in the evaluation of a new compound as a potentialtherapeutic agent. Common targets include enzymes and receptors as wellas nucleic acid ligands displaying characteristic secondary structure.

The emerging paradigm for lead discovery in pharmaceutical and relatedindustries such as agricultural biotechnology, is the assembly of novelsynthetic compound libraries by a broad variety of new methods of solidstate “combinatorial” synthesis. Combinatorial chemistry refers to acategory of strategies for the parallel synthesis and testing ofmultiple compounds or compound mixtures in solution or on solidsupports. For example, a combinatorial synthesis of a linearoligopeptide containing n amino acids would simultaneously create allcompounds representing the possible sequence permutations of n aminoacids. The most commonly employed implementation of combinatorialsynthesis relies on colloidal bead supports to encode reaction steps andthus the identity of each compound. Beads preferred in current practicetend to be large (up to 500 microns in diameter) and porous to maximizetheir compound storage capacity, and they must be encoded to preservethe identity of the compound they carry.

Several methods of encoding, or binary encoding, of beads are available.Two examples are as follows. First, beads may be labeled with shortoligonucleotides such as the 17-mers typically employed in hybridizationexperiments. The sequence of such short probes may be determined bymicroscale sequencing techniques such as direct Maxam-Gilbert sequencingor mass spectrometry. This encoding scheme is suitable when the taskcalls for screening of libraries of nucleic acid ligands oroligopeptides. Second, members of a combinatorial library may beassociated with chemically inert molecular tags. In contrast to theprevious case, these tag molecules are not sequentially linked. Instead,the sequence of reaction steps is encoded by the formal assignment of abinary code to individual tag molecules and their mixtures that areattached to the bead in each successive reaction step. The tags arereadily identified by standard analytical techniques such as gaschromatography. This general encoding strategy is currently employed inthe synthesis of combinatorial libraries on colloidal beads.

Commercial compound libraries are large, given that even for theaforementioned 17-mer, the number of sequence permutations is 4^17, orapproximately 10^10. However, the high specificity of typical biologicalsubstrate-target interactions implies that the vast majority ofcompounds in the collection will be inactive for any one particulartarget. The task of screening is to select from this large set the fewpotential lead compounds displaying activity in binding or in functionalassays. The principal drug discovery strategy widely applied to naturalcompound libraries in the pharmaceutical industry is to selectindividual compounds from the library at random and subject them to aseries of tests. Systematic screening procedures are thus required toimplement the rapid screening and scoring of an entire library ofsynthetic compounds, in practice often containing on the order of 10^7items.

In current practice, compounds are first cleaved and doted from theirsolid supports and are stoned in microtiter plates. Further samplehandling in the course of screening relies primarily on roboticpipetting and transfer between different containers, typically wells inmicrotiter plates. While robotic workstations represent a step in thedirection of automating the process, they rely on the traditional formatof microliter plates containing 8×12 wells and sample handling bypipetting and thus represent merely an incremental operationalimprovement. A significant additional consideration is the need toconserve reagent and sample by reducing the spatial scale of theanalytical procedures.

The present invention provides a set of operations to realize integratedsample handling and screening procedures for bead-based compoundlibraries in a planar format. This will significantly reduce time andcost due to reagent and sample volumes. The principal advantage of themethods of the present invention is that they provide a large set offundamental operations to manipulate sets of beads in a planar format,permitting the handling of beads between stations in a multistepanalytical procedure.

In particular, as previously described herein, the methods of thepresent invention facilitate the implementation of the followingpertinent procedures: transfer of samples from microliter plates to aplanar electrochemical cell; formation of heterogeneous panels of targetsites adjacent to the substrate surface; solid phase, binding assays;and isolation of specific beads from an array. In addition, thefundamental operations of the present invention provide the means toconcatenate these procedures on the surface of a planar electrode.

As described herein for hybridization assays, several variants arepossible. That is, binding assays may be performed by allowing proteintargets such as enzymes to bind to compounds on the surface of a bead,either in suspension or arranged in a planar array. The common practiceof combinatorial chemistry based on large porous carrier beadsaccommodates the concurrent handling of smaller beads to whose outersurface compounds are anchored via inert chemical spacers. Such smallbeads (up to 10 microns in diameter) are readily manipulated by themethods of the present invention. Large beads are used as labeledcompound storage containers.

Alternatively, binding between target and a radioactively or otherwiselabelled probe may occur in solution, within microtiter plate wells, ifcompounds have already been cleaved from their synthesis support. Inthat case, probe-target complexes may be captured by complexation toencoded beads in each well, for example via the secondary antibodymethod of coupling the protein target to a bead-anchored antibody.Bead-captured probe-target complexes are then transferred to the planarcell for proximity analysis and further processing as illustrated inFIG. 10. As shown in FIG. 10, probe-target complexes 102 are allowed toform in solution. Antibody coated beads 104 are added to the solution,resulting in a bead anchored complex 106. The bead anchored complexes106 are deposited onto electrode 108 from wells 110, and a planar arrayof bead anchored complexes is formed. When fluorescent probes 114 areused, these impart fluorescence to the bead anchored complex,facilitating detection.

The methods and apparatus of the present invention are well suited tothe task of identifying a small number of positive events in a largeset. The imaging of an entire array of probe-target complexes is furtherenhanced by proximity to an area detector, and by bead lensing action.The isolation of a small number of positive scores from the array isreadily achieved, for example by applying optical tweezers, as describedherein. The large remainder of the array may then be discarded. This inturn considerably reduces the complexity of applying more stringenttests, such as the determination of binding constants, because these maybe restricted to the few retained beads. These tests may be directlyapplied, without the need for additional sample transfer to newcontainers, to the samples surviving the first screening pass.

Example IX Hybridization Assays in Planar Array Format

The present invention can be used to implement solid phase hybridizationassays in a planar array format in a configuration related to that of aprotein binding assay in which target molecules are chemically attachedto colloidal beads. The methods of the present invention facilitate theformation of a planar array of different target oligonucleotides forpresentation to a mixture of strands in solution. Alternatively, thearray may be formed subsequent to hybridization in solution tofacilitate detection and analysis of the spatial distribution offluorescence or radioactivity in the array.

Considerable research and development is presently being invested in aneffort to develop miniaturized instrumentation for DNA sample extractionand preparation including amplification, transcription, labeling andfragmentation, with subsequent analysis based on hybridization assays aswell as electrophoretic separation. Hybridization assays in planar arrayformat are being developed as a diagnostic tool for the rapid detectionof specific single be pair mutations in a brown segment of DNA, and forthe determination of expression levels of cellular genes via analysis ofthe levels of corresponding mRNAs or cDNAs. Hybridization of twocomplementary single strands of DNA involves molecular recognition andsubsequent hydrogen bond formation between corresponding nucleobases inthe two opposing strands according to the rules A-T and G-C; here A, T,G and C respectively represent the four nucleobases Adenine, Thymine,Guanosine and Cytosine found in DNA; in RNA, Thymine is replaced byUracil. The formation of double-strand, or duplex, DNA requires thepairing of two highly negatively charged strands of DNA, and the ionicstrength of the buffer, along with temperature, plays a decisive role.

As previously discussed herein, two principal methods to prepareheterogeneous arrays of target strands on the surface of a planarsubstrate are micro-dispensing (“printing”) and in-situ, spatiallyencoded synthesis of oligonucleotides representing all possible sequencepermutations for a given total length of strand. In this context,hybridization must necessarily occur in close proximity to a planarsubstrate surface and this condition requires care if complications fromsteric hindrance and from nonspecific binding of strands to thesubstrate are to be avoided. Nonspecific adsorption can be a seriousproblem, especially in the presence of DC electric fields employed incurrent commercial designs that rely on electrophoretic deposition toaccelerate the kinetics of hybridization on the surface. In addition,there are the technical difficulties, previously discussed herein,resulting from steric hindrance and from collective effects reflectingthe crowding of probe strands near the surface.

In the context of DNA analysis, colloidal (magnetic) beads are commonlyused. For example, they are employed to capture DNA in a widely usedscreening procedure to select cDNAs from clone libraries. Specifically,cDNAs are allowed to hybridize to sequences within long genomic DNA thatis subsequently anchored to magnetic beads to extract the hybridizedcDNA from the mixture.

The present invention facilitates the formation of planar arrays ofoligonucleotide-decorated colloidal beads, either prior to or subsequentto hybridization of a fluorescence probe strand to the bead-anchoredtarget strand or subsequent to hybridization in free solution and beadcapture of the end-functionalized target strand. In contrast to priorart methods, the present invention does not require hybridization tooccur in the vicinity of planar substrate surface, although this is anoption if bead-anchored probe strands are to be delivered tosubstrate-anchored target strands.

The ability to perform hybridization either in solution, on the surfaceof individual beads, or at the substrate surface provides anunprecedented degree of flexibility. In addition, the advantages of beadarrays, as described herein, make it feasible to select and isolateindividual beads, or groups of beads, from a larger array on the basisof the score in a hybridization assay. This isolation facilitates theimplementation of subsequent assays on the strands of interest. The factthat beads remain mobile also means that beads of interest may becollected in designated holding areas for microsequencing, or may bemoved to an area of substrate designated for PCR amplification.

The methods of the present invention may be used to implement ahybridization assay in a planar array format in one of two principalvariations. All involve the presence of the entire repertoire of beadsin the planar array or panel formed adjacent to the electrode surfacefor parallel read-out. As with heterogeneous panels in general, thearrangement of beads within the array is either random (with respect tochemical identity), and the identity of beads scoring high in thebinding assay must be determined subsequently, or it is spatiallyencoded by invoking the “Layout-Preserving Transfer” method of sampleloading described herein.

The former variant is readily implemented and accommodates arrayformation either prior to or subsequent to performing the binding assay.For example, binding may be performed in suspension before beads areassembled into the array. As with the aforementioned cDNA selectionprocedure, the method of the present invention also accommodates the useof beads as capture elements for end-functionalized target DNA, forexample, via biotin-streptavidin complexation. In this latter case,beads serve as a delivery vehicle to collect all probe-target complexesto the electrode surface where they are assembled into an array for easeof analysis. In particular, proximity CCD detection of beads onelectrodes will benefit from the lensing action of the beads in thearray. This version of the assay is preferably used if only a smallnumber of positive scores are expected.

Hybridization to a pre-formed bead array can take advantage of a variantof the assay which preserves spatial encoding. An array of bead clustersis formed by the “Layout-Preserving Transfer” method previouslydescribed herein, and exposed to a mixture of cDNAs. The resultingspatial distribution of fluorescence intensity or radioactivity reflectsthe relative abundance of cDNAs in the mixture. This procedure relies onthe detection of a characteristic fluorescence or other signal from theprobe-target complex on the surface of a single bead. Given the factthat the array is readily held stationary by the methods of the presentinvention, image acquisition may be extended to attain robustsignal-to-noise for detection of low level signals. For example, asignal generated by a bead of 10 micron diameter with at most 10^8probe-target complexes on the surface of the bead may be detected. Beadlensing action also aids in detection.

As with the implementation of drug screening, the functional elements ofthe present invention may be combined to perform multiple preparativeand analytical procedures on DNA.

Example X Alignment and Stretching of DNA in Electric Field-Induced Flow

The present invention can be used to position high-molecular weight DNAin its coiled configuration by invoking the fundamental operations asthey apply to other colloidal particles. However, in addition, theelectrokinetic flow induced by an electric field at a patternedelectrode surface may be employed to stretch out the DNA into a linearconfiguration in the direction of the flow.

Procedures have been recently introduced which rely on optical imagingto construct a map of cleavage sites for restriction enzymes along thecontour of an elongated DNA molecule. This is generally known as a“restriction map”. These procedures, which facilitate the study of theinteraction of these and other proteins with DNA and may also lead tothe development of techniques of DNA sequencing, depend on the abilityto stretch and align DNA on a planar substrate.

For individual DNA molecules, this has been previously achieved bysubjecting the molecule to elongational forces such as those exerted byfluid flow, magnetic fields acting on DNA-anchored magnetic beads orcapillary forces. For example, DNA “combs” have been produced by simplyplacing DNA molecules into an evaporating droplet of electrolyte. Ifprovisions are made to promote the chemical attachment of one end of themolecule to the surface, the DNA chain is stretched out as the recedingline of contact between the shrinking droplet and the surface passesover the tethered molecules. This leaves behind dry DNA molecules thatare attached in random positions within the substrate area initiallycovered by the droplet, stretched out to varying degrees and generallyaligned in a pattern of radial symmetry reflecting the droplet shape.Linear “brushes”, composed of a set of DNA molecules chemically tetheredby one end to a common line of anchoring points, have also beenpreviously made by aligning and stretching DNA molecules bydielectrophoresis in AC electric fields applied between two metalelectrodes previously evaporated onto the substrate.

The present invention invokes electrokinetic flow adjacent to anelectrode patterned by UV-mediated regrowth of oxide to provide a novelapproach to the placement of DNA molecules in a predeterminedarrangement on a planar electrode surface, and to the stretching of themolecules from their native coil configuration into a stretched, linearconfiguration that is aligned in a pre-determined direction. Thisprocess is shown in FIG. 11 and is accomplished by creating controlledgradients in the flow vicinity across the dimension of the DNA coil. Thevelocity gradient causes different portions of the coil to move atdifferent velocities thereby stretching out the coil. By maintaining astagnation point at zero velocity, the stretched coil will be fixed inposition. This method has several advantages over the prior artapproaches. First, DNA molecules in their coiled state are subjected tolight control to form arrays of desired shape in any position on thesurface. This is possible because large DNA from cosmids or YACs formscoils with a radius in the range of one micron, and thus acts in amanner analogous to colloidal heads. A set of DNA molecules may thus besteered into a desired initial arrangement. Second, UV-patterningensures that the elongational force created by the electrokinetic flowis directed in a predetermined direction. The presence of metalelectrodes in contact with the sample, a disadvantage of thedielectrophoretic prior art method, is avoided by eliminating thissource of contamination that is difficult to control especially in thepresence of an electric field. On patterned Si/SiOx electrodes, flowvelocities in the range of several microns/second have been generated,as required for the elongation of single DNA molecules in flow. Thus,gradients in the flow field determines both the fractional elongationand the orientation of the emerging linear configurations. Third, thepresent invention facilitates direct, real-time control of the velocityof the electric field-induced flow, and this in turn conveys explicitcontrol over the fractional elongation.

While the invention has been particularly shown and described withreference to a preferred embodiment thereof, it will be understood bythose skilled the art that various changes in form and details may bemade therein without departing from the spirit and scope of theinvention.

What is claimed is:
 1. A method of making a bead array comprising: a)contacting a substrate having a planar surface with a suspensioncomprising populations of several differently-encoded beads havingdiameters of up to 10 microns, wherein each population ofdifferently-encoded beads has a different biomolecule attached theretoand the beads comprise hydrophilic beads; and b) applying energy to saidsubstrate or said suspension, or both, such that the populations ofdifferently-encoded beads form an ordered array on the planar surfaceand the beads are at pre-determined distances from each other.
 2. Themethod according to claim 1 wherein the biomolecule is a protein or anoligonucleotide.
 3. The method according to claim 1 wherein the beadsare immobilized to the substrate following step b).
 4. The methodaccording to claim 3 wherein the beads are immobilized by anchoring tothe substrate.
 5. The method according to claim 3 wherein the beads areimmobilized by chemical attachment, heterobifunctional cross-linkingagents, or physical attraction to the substrate.
 6. The method accordingto claim 5 wherein physical attraction is through van der Waals forces.7. The method according to claim 1 wherein the beads are in a planarcrystalline arrangement.
 8. The method according to claim 1 wherein thebeads are in a hexagonally crystalline configuration.
 9. The methodaccording to claim 1 wherein the beads are in a bubble raft arrangement.10. The method according to claim 1 wherein the beads are encoded usingoligonucleotides.
 11. The method according to claim 1 wherein the beadsare binary encoded.
 12. The method according to claim 1 wherein thesubstrate has a patterned surface.
 13. The method according to claim 1wherein the ordered array of beads comprises subarrays of beads.
 14. Themethod according to claim 1 wherein the beads have a 2-20 microndiameter.
 15. The method according to claim 1 wherein the beads range insize from approximately 100 Angstroms to 10 microns.
 16. The methodaccording to claim 1 wherein the energy is a magnetic field.
 17. Themethod according to claim 1 wherein the energy is electrical energy. 18.The method according to claim 1 wherein the beads are magnetic.
 19. Themethod according to claim 1 wherein the beads comprise hydrophilic beadsand hydrophobic beads.
 20. The method according to claim 19 wherein thebeads are the same size.
 21. The method according to claim 1 wherein thebeads are the same size.